Recent landmark discoveries demonstrate that specific co- activators, like p55/Gcn5p, act as enzymes which facilitate gene transcription by modulating chromatin structure through the acetylation of core histone amino-terminal domains. Evidence now shows that p55/Gcn5p is extensively phosphorylated itself in vivo in Tetrahymena and yeast and that such modifications regulate this enzymes activity in vitro. We propose to investigate the functional role of phosphorylation in regulating p55/Gcn5p histone acetyltransferase (HAT) activity in vivo. One goal is to clearly establish to what extent p55/Gcn5p is phosphorylated in vivo. To this end, we will optimize electrophoretic separation conditions to clearly separate the various isoforms of p55/Gcn5p. This electrophoretic assay will then be used to monitor the phosphorylation status of p55/Gcn5p throughout the life and/or cell cycle of Tetrahymena and yeast as well as during Gcn5p- dependent activated transcription. A second goal is to map the major sites of phosphorylation in p55/Gcn5p and determine how these modifications affect enzyme activity in vitro and in vivo. Phosphorylated sites in p55/Gcn5p will be identified by standard peptide mapping techniques and then mutated by site-directed mutagenesis. These proteins will be expressed and analyzed for HAT activity in vitro. More importantly, these alleles will be transformed into p55/Gcn5p null strains to determine the effects of these mutations in vivo. A third goal will be to identify likely protein kinases responsible for phosphorylation of p55/Gcn5p. This will be accomplished through the use of specific protein kinase inhibitors to block phosphorylation in addition to the use of in-gel kinase assays employing p55/Gcn5p as a substrate to identify kinases from yeast and Tetrahymena extracts. These studies will greatly enhance our understanding of the importance co-activator phosphorylation and may also uncover other post-translational modifications that influence HAT activity or function of this protein.